Our laboratory has recently developed a primary cell culture of guinea gallbladder epithelial cells. The cultured cells form a confluent monolayer, develop transepithelial electrical characteristics and display responses to secretagogues similar to those observed in the native tissue. The cultured cells are suitable for patch clamp techniques and load with and respond appropriately to fluorescent indicator dyes. The present proposal will provide a comprehensive analysis of signaling pathways involved the regulation of gallbladder ion transport by ATP and histamine, agents shown to be important physiologic modulators of cell function in a variety of tissues. Studies are proposed to test the hypothesis that ATP and histamine mediate changes in ion transport by their activation Ca2+ dependent and cAMP-dependent pathways, respectively. Experiments will: 1) determine the purinergic and histamine receptor subtypes initiation the responses; 2) evaluate the role of specific second messenger components in ATP- and histamine-stimulated anion transport reflected as changes in short-circuit current 3) identify the individual ion currents and single channel types underlying changes in Isc to ATP and histamine; 4) determine the role of endogenous intracellular modulators on channel function; and 5) identify possible points of intersection between Ca2+ and ATP-dependent pathways. The results of these studies will provide information concerning signal transduction pathways in the gallbladder and their interaction. This information is relevant to a wide variety of cell types and important to understand the pathophysiology associated with epithelial cell dysfunction.